Compounds for the treatment of Clostridium difficile infection

ABSTRACT

Clostridium difficile  infection (CDI) is a public health threat that results in 14,000 annual deaths in the United States. Challenges involve the production of CDI spores that can remain dormant for years and the production of toxins that damage the gut. Current therapies for CDI include vancomycin and metronidazole, but neither inhibits spore or toxin production. Thus, recurrence of infection occurs in 25% of patients and there are no antibiotics that are effective for multiple recurrences. We describe oxadiazoles with activity against  C. difficile , including the highly virulent NAP1/027 strain with increased production of toxins A and B, as well as the additional binary toxin. Oxadiazole 2 is poorly absorbed, thus advantageously achieving high concentrations in the gut. The compound targets peptidoglycan synthesis and inhibits vegetative cells, spores, and toxin production.

RELATED APPLICATIONS

This application is a National Stage filing under 35 U.S.C. § 371 ofInternational Application No. PCT/US2017/051185 filed Sep. 12, 2017,which claims priority under 35 U.S.C. § 119(e) to U.S. ProvisionalPatent Application No. 62/393,202, filed Sep. 12, 2016, whichapplications are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. AI090818awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Clostridium difficile is a Gram-positive spore-forming anaerobicbacterium that causes life-threatening diarrhea, resulting in 250,000infections per year that require hospitalization and 14,000 annualdeaths in the United States alone. C. difficile infection (CDI) isassociated with the use of antibiotic therapy, where disruption of theintestinal flora allows for C. difficile colonization. C. difficileproduces two toxins, toxin A (TcdA) and toxin B (TcdB), which damageepithelial tissue and promote inflammation that results in rapid fluidloss and diarrhea. Some strains, such as BI/NAP1/027, a highly virulentstrain that has spread widely, have increased production of toxins A andB, as well as the additional binary toxin or CDT. This strain isresponsible for 31% of the hospital-acquired CDIs. The BI/NAP1/027strain has also been described in the community, causing 19% ofcommunity-associated CDIs. The current antibiotics used to treat CDI arevancomycin, metronidazole, and fidaxomicin. Fidaxomicin is a macrolidethat is minimally absorbed, it inhibits RNA synthesis and it has anarrower spectrum of activity for gut microbes. However, treatmentfailure with fidaxomicin is 12% and lower efficacy is observed withfidaxomicin in BI/NAP1/027 CDIs. Metronidazole contains a nitroimidazolegroup that is metabolized to a toxic radical species that damagesbacterial DNA. Metronidazole has high oral bioavailability, resulting inrelative low concentrations in the colon that while approaching MIC arethought to contribute toward resistance development and reduced efficacyin moderate to severe CDI. Vancomycin is poorly absorbed and hasdemonstrated better efficacy than metronidazole, however metronidazoleis used in many countries as first-line therapy because of its low costand due to the risk of vancomycin-resistant enterococci. As a result,vancomycin is recommended only for severe or complicated CDIs.

One of the most challenging issues in management of CDIs is therecurrence. About 25% of patients treated with vancomycin,metronidazole, or fidaxomicin have recurrence of the infection, andthere are no antibiotics that are effective for multiple recurrences.The only option for patients with multiple recurrent CDIs is fecaltransplantation, however safety issues are a concern and fecal donorrecruitment, preparation and administration by nasogastric tube or enemamake it impractical. Another challenge with CDIs is the production ofspores that are dormant for months to years. The spores are notsusceptible to non-chlorine cleaning agents such as detergent orhydrogen peroxide and are insensitive to most antibiotics. Metronidazoleand vancomycin are not active against C. difficile spores at 8× or 80×minimally-inhibitory concentration (MIC). Another difficulty in treatingCDIs is the production of toxins: the enterotoxin TcdA, the cytotoxinTCdB, and the binary toxin CDT. These toxins cause changes that disrupttight junctions and loosen the epithelial barrier, as well as induce therelease of inflammatory mediators, which result in inflammation andneutrophil accumulation that lead to destruction of the intestinalepithelium. Neither vancomycin nor metronidazole inhibit toxinproduction at 8× MIC. Surotomycin, a lipopeptide antibiotic that causesmembrane depolarization, is in phase III clinical trials for treatmentof CDI. However, surotomycin is not active against spores nor inhibitstoxin production.

Accordingly, new antibiotics are needed with low oral bioavailabilitythat can achieve high concentrations in the gastrointestinal track. Theoptimal antibiotic for CDIs would reduce C. difficile vegetative cells,as well as inhibit toxins and spores, and at the same time would notencourage microbial resistance or affect the host microbiota. Inaddition, the antibiotic should not cause adverse events in the host. Itis extremely challenging for an antibiotic to meet all these criteria,therefore new antibiotics for treating CDIs are urgently needed.

SUMMARY

The discovery of oxadiazole ND-421 (compound 1) provided a compoundhaving potent activity against methicillin-resistant Staphylococcusaureus (MRSA). ND-421 has 97% oral bioavailability, low clearance, and along terminal half-life of 18.6 hours, and has efficacy in mouse modelsof MRSA infection. ND-421 also has excellent absorption, therefore, itwould not be useful to treat CDIs. Disclosed herein is oxadiazole 2, acompound that is poorly absorbed and inhibits C. difficile vegetativecells, as well as spores and toxin production. We document thatoxadiazole 2 has better efficacy than vancomycin in the mouse C.difficile model of recurrent infection. Oxadiazole 2 and relatedcompounds disclosed herein can therefore be useful for the treatment ofCDI.

Accordingly, this disclosure provides a compound of Formula (I):

wherein

Q and X are each independently O, NH, N(C₁-C₄(alkyl)), CH₂, or —(C═O)—;

G¹ is (C₃-C₈)cycloalkyl;

R¹ is H, OH, SO₂(C₁-C₄(alkyl)), (C₁-C₈)alkyl, or (C₃-C₈)cycloalkyl; and

R² and G² are each independently H, OH, halo, alkoxy, alkyl, amino,nitro, carboxyl, or —(C═O)NH₂; or

R₁ is optionally bonded to R² to form a heterocycle;

wherein (C₁-C₈)alkyl and (C₃-C₈)cycloalkyl are saturated or optionallyunsaturated, and wherein (C₁-C₈)alkyl and (C₃-C₈)cycloalkyl areoptionally substituted with 1-3 substituents, wherein each substituentis independently halogen, oxo, hydroxy, alkoxy, C₁-C₄(alkyl),trifluoromethyl, trifluoromethoxy, or amino;

with the proviso that when G¹ is cyclopentyl, a bromocyclopentenyl, abromocyclohexenyl, or a 4-aminocyclohexyl, R¹ is not H or allyl, R² isnot H, or Q or X are not O, and with the proviso that when R¹ is bondedto R² to form an indole moiety, Q is not O;

or a pharmaceutically acceptable salt or solvate thereof.

Additionally, a method is disclosed for treating a Clostridium difficilebacterial infection comprising administering to a subject in needthereof an effective amount of a compound of Formula (A), wherein thecompound is not substantially absorbed by the gastrointestinal tractwhere the bacteria to be treated is present, and wherein Formula (A) is:

wherein

Q and X are each independently O, NH, N(C₁-C₄(alkyl)), CH₂, or —(C═O)—;

R¹ and G¹ are each independently H, OH, SO₂(C₁-C₄(alkyl)), aryl,heterocycle, (C₁-C₈)alkyl, or (C₃-C₈)cycloalkyl; and

R² and G² are each independently H, OH, halogen, hydroxy, alkoxy, alkyl,amino, nitro, carboxyl, or —(C═O)NH₂; or

R¹ is optionally bonded to R² to form a heterocycle;

wherein (C₁-C₈)alkyl and (C₃-C₈)cycloalkyl are saturated or optionallyunsaturated, and wherein aryl, heterocycle, (C₁-C₈)alkyl and(C₃-C₈)cycloalkyl are optionally substituted with 1-3 substituents,wherein each substituent is independently halogen, oxo, hydroxy, alkoxy,C₁-C₄(alkyl), trifluoromethyl, trifluoromethoxy, or amino; or apharmaceutically acceptable salt or solvate thereof.

The invention provides novel compounds of Formula I-V, intermediates forthe synthesis of compounds of Formula I-V, as well as methods ofpreparing compounds of Formula I-V. The invention also providescompounds of Formula I-V that are useful as intermediates for thesynthesis of other useful compounds. The invention provides for the useof compounds of Formula I-V for the manufacture of medicaments usefulfor the treatment of bacterial infections in a mammal, such as a human.

The invention provides for the use of the compositions described hereinfor use in medical therapy. The medical therapy can be treatingbacterial infections, for example, an infection by a gram-positivespore-forming anaerobic bacterium. The invention also provides for theuse of a composition as described herein for the manufacture of amedicament to treat a bacterial infection in a mammal, for example, C.difficile infection in a human. The medicament can include apharmaceutically acceptable diluent, excipient, or carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention. However, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1A-1C. Oxadiazole 2 inhibits C. difficile vegetative cells, spores,and toxin production. C. difficile ATCC43255 was inoculated with serialtwo-fold dilutions of oxadiazole or metronidazole, and plated after 24(A), 48 (B), and 72 (C) hours for vegetative cells and spore counts. Theculture supernatant was analyzed for TcdA and TcdB toxins by ELISA.

FIG. 2A-2B. Plasma concentration-time curves after a single po and ivadministration of oxadiazole 2 at 20 mg/kg to mice (n=3 per time pointper route of administration) (A). Comparison of area-under-the-curvesgive absolute oral bioavailability F of 3.8% for oxadiazole 2 (B).

FIG. 3. Oxadiazole 2 inhibits peptidoglycan synthesis in C. difficile bymacromolecular synthesis assays. Oxadiazole 2 was incubated at ½, 1, or2× MIC; data shown are for ½ MIC; positive controls were ciprofloxacin(MIC 8 μg/mL) for DNA, linezolid (MIC 2 μg/mL) for protein, andoxacillin (MIC 8 μg/mL) for peptidoglycan. Maximum inhibition foroxadiazole 2 was observed at 60 min incubation for DNA, at 180 min forprotein, and at 120 min for peptidoglycan.

FIG. 4. Oxadiazole 2 is efficacious in a mouse model of recurrent CDI.Mice (n=10 per group) were given antibiotic cocktail (0.4 mg/mLkanamycin, 0.035 mg/mL gentamicin, 850 U/mL colistin, 0.215 mg/mLmetronidazole, and 0.045 mg/mL vancomycin added to the drinking waterfor five days. After two days, mice were administered a single 10 mg/kgintraperitoneal dose of clindamycin. One day later, the mice wereinfected with 10⁵ cfu of C. difficile strain ATCC 43255intragastrically. Oxadiazole 2 was given po at 20 mg/kg/day starting onehour after infection once a day for 5 days; vancomycin was administeredpo at 50 mg/kg/day for 5 days. Survival was monitored for 25 dayspost-infection. Results are the average of two separate studies.

FIG. 5A-5B. Oxadiazole 2 reduces C. difficile cell count and spores infeces. (A) total cell count and (B) spore count after ethanol treatmentof fecal samples. Day 3: n=7 mice in 20 mg/kg oxadiazole 2, 3 mice in 40mg/kg oxadiazole 2, 7 mice in vancomycin, and 5 in vehicle; Day 7; n=8in 20 mg/kg oxadiazole 2, 6 in 40 mg/kg oxadiazole 2, 4 animals invancomycin group, and 4 in vehicle; Day 10: n=8 in 20 mg/kg oxadiazole2, 7 in 40 mg/kg oxadiazole 2, 5 in vancomycin; no samples obtained invehicle group on day 10.

DETAILED DESCRIPTION

Clostridium difficile infection is an urgent public health threat thatresults in 14,000 annual deaths in the United States alone. The highrecurrence rate and the inability of current antibiotics to treatmultiple recurrent infections requires urgent need for new therapeutics.We describe the discovery of new oxadiazoles that inhibit vegetativecells, spores, and toxin production. The compounds are poorly absorbedand achieve high concentrations in the gut. The oxadiazoles show betterefficacy than vancomycin in a mouse model of recurrent C. difficileinfection.

Definitions

The following definitions are included to provide a clear and consistentunderstanding of the specification and claims. As used herein, therecited terms have the following meanings. All other terms and phrasesused in this specification have their ordinary meanings as one of skillin the art would understand. Such ordinary meanings may be obtained byreference to technical dictionaries, such as Hawley's Condensed ChemicalDictionary 14^(th) Edition, by R. J. Lewis, John Wiley & Sons, New York,N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,etc., indicate that the embodiment described may include a particularaspect, feature, structure, moiety, or characteristic, but not everyembodiment necessarily includes that aspect, feature, structure, moiety,or characteristic. Moreover, such phrases may, but do not necessarily,refer to the same embodiment referred to in other portions of thespecification. Further, when a particular aspect, feature, structure,moiety, or characteristic is described in connection with an embodiment,it is within the knowledge of one skilled in the art to affect orconnect such aspect, feature, structure, moiety, or characteristic withother embodiments, whether or not explicitly described.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds, so that acompound X includes a plurality of compounds X. It is further noted thatthe claims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely,” “only,” and the like, inconnection with any element described herein, and/or the recitation ofclaim elements or use of “negative” limitations.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrases “one or more” and “at least one” are readily understood by oneof skill in the art, particularly when read in context of its usage. Forexample, the phrase can mean one, two, three, four, five, six, ten, 100,or any upper limit approximately 10, 100, or 1000 times higher than arecited lower limit. For example, one or more substituents on a phenylring refers to one to five, or one to four, for example if the phenylring is disubstituted.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements. Whenvalues are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value without themodifier “about” also forms a further aspect.

The terms “about” and “approximately” are used interchangeably. Bothterms can refer to a variation of ±5%, ±10%, ±20%, or ±25% of the valuespecified. For example, “about 50” percent can in some embodiments carrya variation from 45 to 55 percent, or as otherwise defined by aparticular claim. For integer ranges, the term “about” can include oneor two integers greater than and/or less than a recited integer at eachend of the range. Unless indicated otherwise herein, the terms “about”and “approximately” are intended to include values, e.g., weightpercentages, proximate to the recited range that are equivalent in termsof the functionality of the individual ingredient, composition, orembodiment. The terms “about” and “approximately” can also modify theend-points of a recited range as discussed above in this paragraph.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges recited herein also encompass any and all possible sub-ranges andcombinations of sub-ranges thereof, as well as the individual valuesmaking up the range, particularly integer values. It is thereforeunderstood that each unit between two particular units are alsodisclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and14 are also disclosed, individually, and as part of a range. A recitedrange (e.g., weight percentages or carbon groups) includes each specificvalue, integer, decimal, or identity within the range. Any listed rangecan be easily recognized as sufficiently describing and enabling thesame range being broken down into at least equal halves, thirds,quarters, fifths, or tenths. As a non-limiting example, each rangediscussed herein can be readily broken down into a lower third, middlethird and upper third, etc. As will also be understood by one skilled inthe art, all language such as “up to”, “at least”, “greater than”, “lessthan”, “more than”, “or more”, and the like, include the number recitedand such terms refer to ranges that can be subsequently broken down intosub-ranges as discussed above. In the same manner, all ratios recitedherein also include all sub-ratios falling within the broader ratio.Accordingly, specific values recited for radicals, substituents, andranges, are for illustration only; they do not exclude other definedvalues or other values within defined ranges for radicals andsubstituents. It will be further understood that the endpoints of eachof the ranges are significant both in relation to the other endpoint,and independently of the other endpoint.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, theinvention encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group. Additionally, for all purposes, the invention encompassesnot only the main group, but also the main group absent one or more ofthe group members. The invention therefore envisages the explicitexclusion of any one or more of members of a recited group. Accordingly,provisos may apply to any of the disclosed categories or embodimentswhereby any one or more of the recited elements, species, orembodiments, may be excluded from such categories or embodiments, forexample, for use in an explicit negative limitation.

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity, including at the cellularor molecular level, for example, to bring about a physiologicalreaction, a chemical reaction, or a physical change, e.g., in asolution, in a reaction mixture, in vitro, or in vivo.

An “effective amount” refers to an amount effective to treat a disease,disorder, and/or condition, or to bring about a recited effect. Forexample, an effective amount can be an amount effective to reduce theprogression or severity of the condition or symptoms being treated.Determination of a therapeutically effective amount is well within thecapacity of persons skilled in the art. The term “effective amount” isintended to include an amount of a compound described herein, or anamount of a combination of compounds described herein, e.g., that iseffective to treat or prevent a disease or disorder, or to treat thesymptoms of the disease or disorder, in a host. Thus, an “effectiveamount” generally means an amount that provides the desired effect.

The terms “treating”, “treat” and “treatment” include (i) inhibiting thedisease, pathologic or medical condition or arresting its development;(ii) relieving the disease, pathologic or medical condition; and/or(iii) diminishing symptoms associated with the disease, pathologic ormedical condition. Thus, the terms “treat”, “treatment”, and “treating”include lowering, stopping, or reversing the progression or severity ofthe condition or symptoms being treated. As such, the term “treatment”can include medical, therapeutic, and/or prophylactic administration, asappropriate.

The terms “inhibit”, “inhibiting”, and “inhibition” refer to theslowing, halting, or reversing the growth or progression of a disease,infection, condition, or group of cells. The inhibition can be greaterthan about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, comparedto the growth or progression that occurs in the absence of the treatmentor contacting.

The term “substantially” as used herein, is a broad term and is used inits ordinary sense, including, without limitation, being largely but notnecessarily wholly that which is specified. For example, the term couldrefer to a numerical value that may not be 100% the full numericalvalue. The full numerical value may be less by about 1%, about 2%, about3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about10%, about 15%, or about 20%.

In general, a “substituent” refers to an organic group as defined hereinin which one or more bonds to a hydrogen atom contained therein arereplaced by one or more bonds to a non-hydrogen atom such as, but notlimited to, a halogen (i.e., F, Cl, Br, and I); an oxygen atom in groupssuch as hydroxyl groups, alkoxy groups, aryloxy groups, arylalkyloxygroups, oxo(carbonyl) groups, carboxyl groups including carboxylicacids, carboxylates, and carboyxlate esters; a sulfur atom in groupssuch as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups,sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atomin groups such as amines, hydroxylamines, nitriles, nitro groups,N-oxides, hydrazides, azides, and enamines; and other heteroatoms invarious other groups. Non-limiting examples of substituents that can bebonded to a substituted carbon (or other) atom include F, Cl, Br, I,OR′, OC(O)N(R′)₂, CN, CF₃, OCF₃, R′, O, S, C(O), S(O), methylenedioxy,ethylenedioxy, N(R′)₂, SR′, SOR′, SO₂R′, SO₂N(R′)₂, SO₃R′, C(O)R′,C(O)C(O)R′, C(O)CH₂C(O)R′, C(S)R′, C(O)OR′, OC(O)R′, C(O)N(R′)₂,OC(O)N(R′)₂, C(S)N(R′)₂, (CH₂)₀₋₂NHC(O)R′, N(R′)N(R′)C(O)R′,N(R′)N(R′)C(O)OR′, N(R′)N(R′)CON(R′)₂, N(R′)SO₂R′, N(R′)SO₂N(R′)₂,N(R′)C(O)OR′, N(R′)C(O)R′, N(R′)C(S)R′, N(R′)C(O)N(R′)₂,N(R′)C(S)N(R′)₂, N(COR′)COR′, N(OR′)R′, C(═NH)N(R′)₂, C(O)N(OR′)R′, orC(═NOR′)R′ wherein R′ can be hydrogen or a carbon-based moiety, andwherein the carbon-based moiety can itself be further substituted.

“Halo” or “halogen” includes fluoro, chloro, bromo, and iodo.

The terms “carbocyclic” and “carbocycle” denote a ring structure whereinthe atoms of the ring are carbon. In some embodiments, the carbocyclehas 3 to 8 ring members, whereas in other embodiments the number of ringcarbon atoms is 4, 5, 6, or 7.

The term “alkoxy” refers to the group alkyl-O—, where alkyl is asdefined herein. Examples of alkoxy groups include, but are not limitedto, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy,sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like. Thealkoxy can be unsubstituted or substituted as described for alkylgroups.

The term “amine” includes primary, secondary, and tertiary amineshaving, e.g., the formula N(group)₃ wherein each group can independentlybe H or non-H, such as alkyl, aryl, and the like. Amines include but arenot limited to R—NH₂, for example, alkylamines, arylamines,alkylarylamines; R₂NH wherein each R is independently selected, such asdialkylamines, diarylamines, aralkylamines, heterocyclylamines and thelike; and R3N wherein each R is independently selected, such astrialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, andthe like. The term “amine” also includes ammonium ions as used herein.

An “amino” group is a substituent of the form —NH₂, —NHR, —NR₂, —NR₃ ⁺,wherein each R is an independently selected substituent such as alkyl,optionally including protonated forms of each. Accordingly, any compoundsubstituted with an amino group can be viewed as an amine.

The term “aryl” refers to an aromatic hydrocarbon group derived from theremoval of at least one hydrogen atom from a single carbon atom of aparent aromatic ring system. The radical attachment site can be at asaturated or unsaturated carbon atom of the parent ring system. The arylgroup can have from 6 to 30 carbon atoms, for example, about 6-10 carbonatoms. The aryl group can have a single ring (e.g., phenyl) or multiplecondensed (fused) rings, wherein at least one ring is aromatic (e.g.,naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Typical arylgroups include, but are not limited to, radicals derived from benzene,naphthalene, anthracene, biphenyl, and the like. The aryl can beunsubstituted or optionally substituted, as described for alkyl groups(below).

The term “heterocycle” refers to a saturated or partially unsaturatedring system, containing at least one heteroatom selected from the groupoxygen, nitrogen, silicon, and sulfur, and optionally substituted withone or more groups as defined for the term “substituted”. A heterocyclecan be a monocyclic, bicyclic, or tricyclic group. Such heterocycles mayalso be aromatic. Therefore, “heteroaryls” are a subset of heterocycles.A heterocycle group also can contain an oxo group (═O) or a thioxo (═S)group attached to the ring. Non-limiting examples of heterocycle groupsinclude 1,3-dihydrobenzofuran, 1,3-dioxolane, 1,4-dioxane, 1,4-dithiane,2H-pyran, 2-pyrazoline, 4H-pyran, chromanyl, imidazolidinyl,imidazolinyl, indolinyl, isochromanyl, isoindolinyl, morpholinyl,piperazinyl, piperidinyl, pyrazolidinyl, pyrazolinyl, pyrrolidine,pyrroline, quinuclidine, tetrahydrofuranyl, and thiomorpholine.

The term “heteroaryl” refers to a monocyclic, bicyclic, or tricyclicring system containing one, two, or three aromatic rings and containingat least one nitrogen, oxygen, or sulfur atom in an aromatic ring. Theheteroaryl can be unsubstituted or substituted, for example, with one ormore, and in particular one to three, substituents, as described in thedefinition of “substituted”. Typical heteroaryl groups contain 2-20carbon atoms in the ring skeleton in addition to the one or moreheteroatoms. Examples of heteroaryl groups include, but are not limitedto, 2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridinyl,benzo[b]thienyl, benzothiazolyl, β-carbolinyl, carbazolyl, chromenyl,cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl,imidizolyl, indazolyl, indolisinyl, indolyl, isobenzofuranyl,isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl,oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl,phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl,pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, tetrazolyl,and xanthenyl.

A “salt” as is well known in the art includes an organic compound suchas a carboxylic acid, a sulfonic acid, or an amine, in ionic form, incombination with a counterion. For example, acids in their anionic formcan form salts with cations such as metal cations, for example sodium,potassium, and the like; with ammonium salts such as NH₄ ⁺ or thecations of various amines, including tetraalkyl ammonium salts such astetramethylammonium, or other cations such as trimethylsulfonium, andthe like. A “pharmaceutically acceptable” or “pharmacologicallyacceptable” salt is a salt formed from an ion that has been approved forhuman consumption and is generally non-toxic, such as a chloride salt ora sodium salt. A “zwitterion” is an internal salt such as can be formedin a molecule that has at least two ionizable groups, one forming ananion and the other a cation, which serve to balance each other. Forexample, amino acids such as glycine can exist in a zwitterionic form. A“zwitterion” is a salt within the meaning herein.

Embodiments of the Invention

Various embodiments herein disclose a compound of Formula (I):

wherein

Q and X are each independently O, NH, N(C₁-C₄(alkyl)), CH₂, or —(C═O)—;

G¹ is (C₃-C₈)cycloalkyl;

R¹ is H, OH, SO₂(C₁-C₄(alkyl)), (C₁-C₈)alkyl, or (C₃-C₈)cycloalkyl; and

R² and G² are each independently H, OH, halo, alkoxy, alkyl, amino,nitro, carboxyl, or —(C═O)NH₂; or

R¹ is optionally bonded to R² to form a heterocycle, where Q isoptionally a direct bond and —R¹—R²— forms a 5-6 membered optionallyunsaturated ring containing one, two, or three heteroatoms;

wherein (C₁-C₈)alkyl and (C₃-C₈)cycloalkyl are saturated or optionallyunsaturated, and wherein (C₁-C₈)alkyl and (C₃-C₈)cycloalkyl areoptionally substituted with 1-3 substituents, wherein each substituentis independently halogen, oxo, hydroxy, alkoxy, C₁-C₄(alkyl),trifluoromethyl, trifluoromethoxy, or amino;

or a pharmaceutically acceptable salt or solvate thereof.

In various embodiments, Formula (I) includes the proviso that when G¹ iscyclopentyl, a bromocyclopentenyl, a bromocyclohexenyl, or a4-aminocyclohexyl, R¹ is not H or allyl, R² is not H, or Q or X are notO. In further embodiments, Formula (I) can include the proviso that whenR¹ is bonded to R² to form an indole moiety, Q is not O. Furthermore,any one or more elements recited for a formula herein may also beexplicitly excluded from an embodiment to more particularly claim thescope of the invention.

Various embodiments of alkyl groups include straight chain and branchedalkyl groups and cycloalkyl groups having from 1 to about 20 carbonatoms, and typically from 1 to 12 carbons or, in some embodiments, from1 to 8 carbon atoms. Examples of straight chain alkyl groups includethose with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl,n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples ofbranched alkyl groups include, but are not limited to, isopropyl,iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and2,2-dimethylpropyl groups. Representative substituted alkyl groups canbe substituted one or more times with any of the groups listed above, orfor example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, andhalogen groups.

Various embodiments of cycloalkyl groups are cyclic alkyl groups suchas, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, thecycloalkyl group can have 3 to about 8-12 ring members, whereas in otherembodiments the number of ring carbon atoms range from 3 to 5, 6, or 7.Cycloalkyl groups further include polycyclic cycloalkyl groups such as,but not limited to, norbornyl, adamantyl, bornyl, camphenyl,isocamphenyl, and carenyl groups, and fused rings such as, but notlimited to, decalinyl, and the like. Cycloalkyl groups also includerings that are substituted with straight or branched chain alkyl groupsas defined above. Representative substituted cycloalkyl groups can bemono-substituted or substituted more than once, such as, but not limitedto, 2,2-, 2,3-, 2,4-, 2,5- or 2,6-disubstituted cyclohexyl groups ormono-, di- or tri-substituted norbornyl or cycloheptyl groups, which canbe substituted with, for example, amino, hydroxy, cyano, carboxy, nitro,thio, alkoxy, and halogen groups.

In some embodiments, Formula (I) is a compound of Formula (II):

The group Q can be ortho, meta, or para to the bond of its phenyl ringto the oxadiazole moiety. Furthermore, the group R² can be ortho, meta,or para to the bond of its phenyl ring to the oxadiazole moiety providedthat Q is not in that same position.

In other embodiments, Formula (I) is a compound of Formula (III):

The group X can be ortho, meta, or para to the bond of its phenyl ringto the oxadiazole moiety. Furthermore, the group G² can be ortho, meta,or para to the bond of its phenyl ring to the oxadiazole moiety providedthat X is not in that same position.

In yet other embodiments, Formula (I) is a compound of Formula (IV):

In various embodiments, the groups R² and G² can be ortho or meta to thebond of its phenyl ring to the oxadiazole moiety.

In additional embodiments of Formula (I) is a compound of Formula (V):

In various embodiments of the disclosed formulas, G² is H. In otherembodiments R² is H. In yet other embodiments, R² is H and G² is H. Insome embodiments X is O and G¹ is cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, or cycloheptyl. In additional embodiments, R¹ is bonded toR² to form a 5- or 6-membered heteroaryl moiety. Examples include, butare not limited to, a benzimidazole, a benzofuran, a benzotriazole, or abenzoxazole. In various other embodiments, a compound of the disclosedformulas is an optically active compound (i.e., scalemic orsubstantially optically pure). In other embodiments of the disclosedformulas, Q is N, NH, N(C₁-C₄(alkyl)), C, CH, CH₂, or —(C═O)—. Forexample, when R¹ is bonded to R² and Q is N, the moiety formed can be aquinoline, or if Q is CH, the moiety formed can be a naphthalene. Inanother example. If Q is C and R¹ is an unsaturated alkyl, thesubstituent formed can be an acetylene. In some particular embodiments,the compound of Formula (I) can be one of compounds i-viii:

In various embodiments, a pharmaceutical composition comprises anycompound disclosed herein, or comprises a compound of any of FormulasI-V, and a pharmaceutically acceptable diluent or carrier.

Additionally, this disclosure provides a method of treating aClostridium difficile bacterial infection comprising administering to asubject in need thereof an effective amount of a compound of Formula(A), wherein the compound is not substantially absorbed (or issubstantially not absorbed) by the gastrointestinal tract where thebacteria to be treated is present, and wherein Formula (A) is:

wherein

Q and X are each independently O, NH, N(C₁-C₄(alkyl)), CH₂, or —(C═O)—;

R¹ and G¹ are each independently H, OH, SO₂(C₁-C₄(alkyl)), aryl,heterocycle, (C₁-C₈)alkyl, or (C₃-C₈)cycloalkyl; and

R² and G² are each independently H, OH, halogen, hydroxy, alkoxy, alkyl,amino, nitro, carboxyl, or —(C═O)NH₂; or

R¹ is optionally bonded to R² to form a heterocycle, where Q isoptionally a direct bond and —R¹—R²— forms a 5-6 membered optionallyunsaturated ring containing one, two, or three heteroatoms;

wherein (C₁-C₈)alkyl and (C₃-C₈)cycloalkyl are saturated or optionallyunsaturated, and wherein aryl, heterocycle, (C₁-C₈)alkyl and(C₃-C₈)cycloalkyl are optionally substituted with 1-3 substituents,wherein each substituent is independently halogen, oxo, hydroxy, alkoxy,C₁-C₄(alkyl), trifluoromethyl, trifluoromethoxy, or amino;

or a pharmaceutically acceptable salt or solvate thereof.

Embodiments of the disclosed methods include compounds of Formula (A)that are compounds of Formula (B):

Embodiments of this disclosure include compounds that are administeredorally (po). In various embodiments for the treatment of a Clostridiumdifficile bacterial infection, less than 30 percent of the compound isabsorbed by the gastrointestinal tract where the bacteria to be treatedis present, and wherein absorption is measured, for example, at 24 hoursafter administration. In other embodiments, less than 25 percent, lessthan 20 percent, less than 15 percent, less than 10 percent, less than 5percent, less than 3 percent, or less than 2.5 percent of the compoundis absorbed by the gastrointestinal tract where the bacteria to betreated is present. The administration can be enteral administration,for example, oral, sublingual, or rectal. An ordinary person skilled inthe art of drug metabolism or pharmacokinetics would, for example,administer a disclosed compound to an animal by po, then take bloodsamples at various time points for analysis of the compound present inthe sample and determine various pharmacokinetic parameters, such as acalculation to determine how much of a disclosed compound was absorbedand how much additional compound should be administered.

In yet other embodiments, the compound is one of compounds i-viii,and/or one of compounds ix-xxii, or any other compound described orillustrated herein:

Embodiments of this disclosure include the use of a compound of Formula(A) for the therapeutic treatment of a Clostridium difficile bacterialinfection, wherein Formula (A) is:

wherein

Q and X are each independently O, NH, N(C₁-C₄(alkyl)), CH₂, or —(C═O)—;

R¹ and G¹ are each independently H, OH, SO₂(C₁-C₄(alkyl)), aryl,heterocycle, (C₁-C₈)alkyl, or (C₃-C₈)cycloalkyl; and

R² and G² are each independently H, OH, halogen, hydroxy, alkoxy, alkyl,amino, nitro, carboxyl, or —(C═O)NH₂, or

R¹ is optionally bonded to R² to form a heterocycle;

wherein (C₁-C₈)alkyl and (C₃-C₈)cycloalkyl are saturated or optionallyunsaturated, and wherein aryl, heterocycle, (C₁-C₈)alkyl and(C₃-C₈)cycloalkyl are optionally substituted with 1-3 substituents,wherein each substituent is independently halogen, oxo, hydroxy, alkoxy,C₁-C₄(alkyl), trifluoromethyl, trifluoromethoxy, or amino;

or a pharmaceutically acceptable salt or solvate thereof.

Various embodiments include the use of any one of the disclosedcompounds, or a compound of the disclosed formulas, for the treatment ofa Clostridium difficile bacterial infection. Other embodiments includethe use of any one of the disclosed compounds for the treatment ofClostridium difficile bacterial infection comprising oral administrationof the compound wherein less than 30 percent, 20 percent, 10 percent, or5 percent of the compound is absorbed by the gastrointestinal tract.Several compounds were also evaluated for activity against C. difficileand were found to be equally or more effective toward C. difficile thanS. aureus, as shown in the table below.

Compound C. difficile MIC vii 8 ix 2 xi 2 xvii 4 xxi 4 xxii 2

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound described orillustrated herein, which possess the useful properties describedherein, it being well known in the art how to prepare optically activeforms (for example, by synthesis from optically-active startingmaterials, by using resolution of the racemic form by recrystallizationtechniques, by chiral synthesis, or by chromatographic separation usinga chiral stationary phase). Thus, the compounds of this inventioninclude all stereochemical isomers arising from the various structuralvariations of these compounds.

Results and Discussion

Synthesis and Activity Profile of Oxadiazole 2

Oxadiazole 2 was synthesized in four steps as shown in Scheme 1. ND-421and oxadiazole 2 was evaluated in 14 C. difficile strains, withvancomycin, metronidazole, and fidaxomycin as controls (Table 1). Theminimally-inhibitory concentration (MIC) ranged from 1-2 μg/mL forND-421 and oxadiazole 2, 0.25-1 μg/mL for vancomycin, 0.1-0.25 μg/mL formetronidazole, and ≤0.01-0.25 for fidaxomicin. The oxadiazoles hadsimilar activity against NAP1/027 strains BAA-1870 and BAA-1803 asvancomycin.

TABLE 1 Minimal-inhibitory concentrations (MICs) of oxadiazoles (μg/mL)at 24 h and 48 h. 1 2 vancomycin metronidazole fidaxomicin C. difficileATCC43598^(a) 1/1 1/2 1/1 0.125/0.125 0.01/0.01 C. difficileATCC43255^(b) 2/2 2/2 0.5/0.5 0.01/0.01 0.01/0.01 C. difficileBAA-1870^(c) 2/4 2/2 1/2 0.25/0.5  <0.25/0.25   C. difficileBAA-1804^(d) 2/2 2/2 1/1 0.25/0.25 <0.25/<0.25 C. difficile BAA-1803^(e)1/2 1/2 1/2 0.25/0.5  0.5/1   C. difficile BAA-1801^(f) 2/4 2/20.25/0.25 0.125/0.125  0.03/0.125 C. difficile BAA-1812^(g) 2/2 2/20.25/0.5  0.25/0.25 0.015/0.125 C. difficile BAA-1814^(h) 2/4 2/20.5/0.5 0.25/0.25 ≤0.01/0.03    C. difficile NR49310^(i) 2/2 2/40.25/0.5  0.125/0.125 ≤0.01/≤0.01 C. difficile NR49294^(j) 1/1 1/20.25/0.25 0.25/0.25 ≤0.01/0.01    C. difficile NR49305^(k) 2/4 2/20.25/0.5  0.25/0.25 0.01/0.01 C. difficile NR49318^(l) 2/2 2/4 0.25/0.250.25/0.25  0.25/0.125 C. difficile NR49292^(m) 1/2 1/2 0.25/0.250.125/0.25  0.25/0.5  C. difficile NR49302^(n) 1/2 2/2 0.25/0.5 0.125/0.125 0.03/0.03 ^(a)isolated from human feces, toxinotype VIII,TcdA− (toxin A), TcdB+ (toxin B) ^(b)isolated from abdominal wound,TcdA+, TcdB+ ^(c)NAP1, BI 8, ribotype 27, toxinotype IIIb, TcdA+, TcdB+,CDT+ (binary toxin) ^(d)toxinotype 0, ribotype 053 TcdA+, TcdB+, CDT+^(e)clinical isolate, NAP1, toxinotype IIIc, ribotype 027, tcdA+, tcdB+,cdtB+ ^(f)isolated from human feces, nontoxigenic, TcdA−, TcdB−,ribotype 010 ^(g)toxinotype XII, ribotype 024, TcdA+, TcdB+, CDT−^(h)toxinotype XXII, ribotype 251, TcdA+, TcdB−, CDT+ ^(i)isolated fromhuman feces, NAP7, ribotype 078, TcdA+, TcdB+, TcdC+, (Δ39), CDT+^(j)isolated from human feces, NAP4, ribotype 014, TcdA+, TcdB+, TcdC+,CDT−, most prevalent after NAP1/027 ^(k)isolated from human feces, NAP6,ribotype 002, TcdA+, TcdB+, TcdC+, CDT−, community associated epidemicstrain ^(l)isolated from human feces, NAP11, ribotype 106, TcdA+, TcdB+,TcdC+, CDT−, predominant epidemic strain in a children's hospital inChicago, increased risk of relapses ^(m)isolated from human feces, NAP2,ribotype 001_072, TcdA+, TcdB+, TcdC+, CDT−, epidemic in US during 1990sand still common ^(n)isolated from human feces, NAP4, ribotype 020,TcdA+, TcdB+, TcdC+, CDT−, among top 7 isolates in 2011-2012

The activity of oxadiazoles 1 and 2 against common gut bacteria wasinvestigated. The oxadiazoles were not active against Lactobacillusreuteri, Lactobacillus gasseri, and Veillonella sp. (Table 2).

TABLE 2 Minimal-inhibitory concentrations (MICs) of oxadiazoles (μg/mL)against common gut bacteria. 1 2 vancomycin metronidazole fidaxomicinBacteroides fragilis HM-709^(a) 2 1 16 0.5 >32 Bacteroides fragilis 4 216 1 >32 Bacteroides ovatus 4 2 16 2 >32 Bacteroides vulgatus 2 2 162 >32 Bacteroides eggerthii 2 1 8 2 >32 Bacteroides caccae 4 2 32 2 >32Bifidobacterium longum HM-846^(b) 0.2 1 0.25 0.5 <0.01 Corynebacteriumsp. HM-784^(c) 2 0.5 0.25 >32 <0.06 Fusobacterium nucleatum HM-992^(d) 12 0.25 2 <0.06 Lactobacillus reuteri HM-102^(e) >12 >128 >32 >32 >32Lactobacillus gasseri HM-644^(f) >12 >128 1 >32 2 Veillonella sp.HM-49^(g) >12 >128 >32 2 8 Eubacterium sp. HM-178^(h) 2 4 2 1 16^(a)Gram-negative, anaerobic bacterium that is commensal and critical tohost immunity; a minor component of the human gut microflora (<1%)^(b)Anaerobic, Gram-positive bacterium commonly found in the normalhuman intestinal microflora ^(c)Isolated from human feces,non-sporulating, Gram-positive, aerobic or facultatively anaerobicbacterium that occurs in the mucosa and normal skin flora of humans andanimals ^(d)Anaerobic, non-sporulating, Gram-negative bacterium commonlyfound in the gastrointestinal tract ^(e)Gram-positive, anaerobicbacteria commonly found in the normal human gastrointestinal tract,commonly used as a probiotic to maintain balance of gut microbial flora^(f)Gram-positive, facultative, anaerobe bacterium commonly found in thenormal human gastrointestinal tract, commonly use in yougurt productionas a probiotic to suppress Helicobacter pylori infections^(g)Gram-negative, non-sporulating bacterium commonly found in theintestinal tract of humans and animals ^(h)Anaerobic, non-sporulating,Gram-positive bacterium commonly found in the gastrointestinal flora ofhumans and animalsOxadiazole 2 Inhibits Vegetative Cells, Spores, and Toxin Production

We evaluated compound 2 for its ability to inhibit C. difficilevegetative cells, spores, and toxin production. At 24 hours, oxadiazole2 inhibited growth of vegetative cells, as well as toxin production at1× MIC (FIG. 1); there were no spores produced. At 48 hours, oxadiazoleinhibited spore production at ¼ MIC and toxin production at 1× MIC; at72 hours, vegetative cells, spores, and toxin production were inhibitedat 1× MIC.

Oxadiazole 2 is Poorly Absorbed

The pharmacokinetics of oxadiazole 2 were investigated after single oral(po) and intravenous (iv) administration to uninfected mice.Concentrations versus time are shown in FIG. 2A and pharmacokineticparameters are summarized in FIG. 2B. After a 20 mg/kg iv dose ofoxadiazole 2, clearance was moderate at 17.4 mL/min/kg (approximately20% of hepatic blood flow), the volume of distribution (V_(d)) of 1.8L/kg was large indicating that the compound distributed to tissues, andthe elimination half-life was 1.2 h. Systemic exposure as measured bythe area-under-the-curve (AUC_(0-∞)) was 1149 μg·min/mL after ivadministration and 44 μg·min/mL following a po dose, giving an absoluteoral bioavailability of 4%. C_(max) of 0.30 μg/mL was attained at 1 hand the elimination half-life was 1.1 h. Feces were collected from themice via metabolism cages and levels of oxadiazole 2 were 43±3 μg/g(equivalent to 43±3 μg/mL assuming a density of 1 g/mL) or about 20× MICat 24 h after a single 20 mg/kg po dose. For in vivo efficacy against C.difficile, antibiotics with poor oral bioavailability that are poorlyabsorbed (or not absorbed) are needed for treatment of this difficultpathogen in the gastrointestinal tract. Oxadiazole 2 and relatedoxadiazoles described herein fit these criteria.

Oral Administration of Oxadiazole 2 Results in High Concentrations inthe Gut and Is Well Tolerated in Mice

We evaluated the toxicity of oxadiazole 2 in uninfected mice aftermultiple-dose oral administration at 40 mg/kg/day for 5 days. We alsocollected feces and analyzed them by ultraperformance liquidchromatography (UPLC) with multiple-reaction monitoring (MRM) detection.Oxadiazole 2 was well tolerated and did not result in weight loss (Table3). On day 5, concentrations of oxadiazole 2 were 30.8±11.1 μg/g(equivalent to 30.8 μg/mL assuming a density of 1 g/mL, Table 3), 15- to30-fold higher than MIC.

TABLE 3 Concentration of oxadiazole 2 in feces after multiple oral doseadministration. Oxadiazole 2 in Day Feces (μg/g feces) Body weight (g) 1 9.8 ± 0.4 19.6 ± 0.6 2 15.0 ± 3.7 19.8 ± 0.6 3 18.1 ± 2.4 19.8 ± 1.0 416.9 ± 1.8 19.7 ± 1.0 5  30.8 ± 11.1 19.4 ± 0.6Oxadiazole 2 Inhibits Cell Wall Synthesis

We investigated the mechanism of action of oxadiazole 2 using themacromolecular synthesis assay with C. difficile ATCC 43255.Incorporation of radiolabeled [2,8-³H]adenine into DNA,1-[3,4,5-³H(N)]-leucine into protein, and N-acetyl-d-[6-³H]-glucosamineinto peptidoglycan (cell wall) was monitored and compared to positivecontrols (ciprofloxacin, linezolid, and oxacillin for DNA, protein, andpeptidoglycan, respectively). Oxadiazole 2 inhibited peptidoglycansynthesis 54.8±3.7% compared to 54.1±13 for the positive controloxacillin (FIG. 3). In contrast, inhibition of DNA or protein byoxadiazole 2 was not significant.

Efficacy of Oxadiazole 2 in a Mouse Model of Recurrent CDI

We next evaluated oxadiazole 2 in the mouse model of recurrent CDIdeveloped by Chen et al. (Gastroenterology 2008, 135(6), 1984). Whileseveral animal models are available to study CDI, the most widely usedanimal model of CDI is the hamster, as the hamster is readilysusceptible to CDI. However, in hamsters primarily the cecum and ileumare affected and animals develop severe enterocolitis and quickly die.As such, the hamster model does not reproduce the typical course ofdisease in humans. The mouse model parallels the human disease in thatthe entire colon is affected. The model results in variable diseaseseverity, which resurges after vancomycin therapy, and is a recurrentdisease in animals that survive the initial infection. Mice were treatedwith a mixture of kanamycin, gentamicin, colistin (850 U/mL),metronidazole, and vancomycin in the drinking water for five days, andclindamycin given intraperitoneally two days later. The following daymice were infected with C. difficile strain ATCC 43255 intragastrically.Oxadiazole 2 was given po at 20 mg/kg/day starting one hour afterinfection once a day for 5 days; vancomycin was administered po at 50mg/kg/day for 5 days. After 25 days, survival was 45% in vancomycin, 70%in oxadiazole 2, and 40% in vehicle (FIG. 3). Fecal pellets or diarrheasmears were collected from the surviving mice and plated for vegetativecell and spore counts, as well as analyzed for TcdA and TcdB toxins byELISA. Compound 2 resulted in about 3-log reduction in vegetative cellsby day 10, 3-log decrease in spores were observed in the 20 mg/kg group,while the 40 mg/kg group showed no spores on days 3, 7, and 10 (FIG.3B). In contrast, vancomycin resulted in 2-log reduction in vegetativecells; no spores were observed until day 10.

Subsequently, colony counts in feces were conducted following treatmentwith oxadizole 2 at 20 or 40 mg/kg/day for 5 days. Total cell countswere 3.44 log lower on day 10 in the 20 mg/kg oxadiazole 2 group and4.68 log lower in the 40 mg/kg group compared to day 3, while thevancomycin-treated group showed 1.02 log reduction (FIG. 5A). Treatmentwith 20 mg/kg oxadiazole 2 resulted in 1.85 log reduction in spores,while no spores were observed at any time in mice given 40 mg/kgoxadiazole 2 (FIG. 5B). Mice treated with 50 mg/kg vancomycin did notproduce spores initially, however once vancomycin treatment stoppedspores were observed on day 10 (FIG. 5B). The vancomycin results areconsistent with those observed by Chen et al. due torecurrence/relapsing CDI after discontinuation of treatment withvancomycin. Likewise, no toxin production was detected in the 40 mg/kgoxadiazole 2 group, while toxin was observed in the vancomycin group onday 10 (Table 4). These studies demonstrate that oxadiazole 2 has invivo efficacy in a mouse model of recurrent CDI.

TABLE 4 TcdA and TcdB toxin detection in C. difficile infected mice. Day3 Day 7 Day 10 Compound 2 (20 mg/kg) + + + Compound 2 (40 mg/kg) − − −Vancomycin (50 mg/kg) − − + Vehicle + +General Synthetic Methods

In general, preparation of the compounds and formulas described herein,and modifications thereof, can be made according to organic synthesistechniques known to those of skill in the art and/or according to thesynthetic schemes provided herein, such as Scheme 1 above. Wheredesired, synthesis of a subject compound can begin with commerciallyavailable chemicals, from compounds described in the chemicalliterature, or from products of the reactions and methods describedherein. Commercially available compounds may be obtained from standardcommercial sources including Acros Organics (Pittsburgh, Pa.), AldrichChemical (Milwaukee, Wis., including Sigma Chemical and Fluka), EastmanOrganic Chemicals, Eastman Kodak Company (Rochester, N.Y.), FisherScientific Co. (Pittsburgh, Pa.), ICN Biomedicals, Inc. (Costa Mesa,Calif.), Lancaster Synthesis (Windham, N.H.), Spectrum Quality Product,Inc. (New Brunswick, N.J.), TCI America (Portland, Oreg.), Combi-Blocks,Inc. (San Diego, Calif.), Oakwood Products, Inc. (Estill, S.C.), andWako Chemicals USA, Inc. (Richmond, Va.).

In addition, methods known to one of ordinary skill in the art may beidentified through various reference books and databases. Suitablereference books and treatises that detail the synthesis of reactantsuseful in the preparation of the inhibiting agents described herein, orprovide references to articles that describe the preparation, includefor example, “Synthetic Organic Chemistry”, John Wiley & Sons, Inc., NewYork; S. R. Sandler et al., “Organic Functional Group Preparations,” 2ndEd., Academic Press, New York, 1983; H. O. House, “Modern SyntheticReactions”, 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L.Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, NewYork, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanismsand Structure”, 4th Ed., Wiley-Interscience, New York, 1992; andProtecting Groups in Organic Synthesis, Second Edition, Greene, T. W.,and Wutz, P. G. M., John Wiley & Sons, New York.

Additional suitable reference books and treatise that detail thesynthesis of reactants useful in the preparation of compounds describedherein, or provide references to articles that describe the preparation,include for example, Fuhrhop, J. and Penzlin G. “Organic Synthesis:Concepts, Methods, Starting Materials”, Second, Revised and EnlargedEdition (1994) John Wiley & Sons ISBN: 3-527-29074-5; Hoffman, R. V.“Organic Chemistry, An Intermediate Text” (1996) Oxford UniversityPress, ISBN 0-19-509618-5; Larock, R. C. “Comprehensive OrganicTransformations: A Guide to Functional Group Preparations” 2nd Edition(1999) Wiley-VCH, ISBN: 0-471-19031-4; March, J. “Advanced OrganicChemistry: Reactions, Mechanisms, and Structure” 4th Edition (1992) JohnWiley & Sons, ISBN: 0-471-60180-2; Patai, S. “Patai's 1992 Guide to theChemistry of Functional Groups” (1992) Interscience ISBN: 0-471-93022-9;Solomons, T. W. G. “Organic Chemistry” 7th Edition (2000) John Wiley &Sons, ISBN: 0-471-19095-0; “Industrial Organic Chemicals: StartingMaterials and Intermediates: An Ullmann's Encyclopedia” (1999) JohnWiley & Sons, ISBN: 3-527-29645-X, in 8 volumes; “Organic Reactions”(1942-2000) John Wiley & Sons, in over 55 volumes; and “Chemistry ofFunctional Groups” John Wiley & Sons, in 73 volumes.

Unless specified to the contrary, the reactions described herein takeplace at atmospheric pressure, generally within a temperature range from−10° C. to 200° C. Further, except as otherwise specified, reactiontimes and conditions are intended to be approximate, e.g., taking placeat about atmospheric pressure within a temperature range of about −10°C. to about 110° C. over a period of about 1 to about 24 hours;reactions left to run overnight average a period of about 16 hours.

A number of exemplary methods for preparation of the compounds of theinvention are provided below. These methods are intended to illustratethe nature of such preparations are not intended to limit the scope ofapplicable methods. Other variations, such as adding varioussubstituents (e.g., as defined above) on various alkyl, cycloalkyl,aryl, or heterocycle groups are included in the scope of the invention.Relevant starting materials can typically be purchased from thecommercial suppliers cited above (e.g., from Sigma-Aldrich, Milwaukee,Wis.) or they can be prepared in a few standard steps from commerciallyavailable materials.

In various embodiments, compounds of the formulas described herein canbe prepared by the following representative methods, as illustrated bySchemes 2-8.

where R and G are substituent groups as defined above, and for example,and recited for R¹ and G¹ in Formulas I and A. As indicated above, R(i.e., —OR) and G can be located at any available position on theirrespective phenyl rings, ortho, meta, or para to the site of attachmentto the oxadiazole moiety. Furthermore, R or G can be an ortho-fusedheterocycle or heteroaryl group, as represented by the respective moietyin one or more of compounds i-xxii. In other embodiments, the oxygenatom of —OR can be replaced with a nitrogen to provide a —NH—Rsubstituent (see Scheme 5). Additional examples of compounds of theinvention and methods for their preparation are provided in the schemesbelow.

As would be readily recognized by one of skill in the art, various othersubstituents (e.g., R¹ and G¹ groups of Formulas A and I) can beinstalled by selection of the appropriate commercially availablestarting material and/or the relevant starting material can be preparedby standard synthetic techniques known to those of skill in the art toprovide the compounds of the formulas described herein.Pharmaceutical Formulations

The compounds described herein can be used to prepare therapeuticpharmaceutical compositions, for example, by combining the compoundswith a pharmaceutically acceptable diluent, excipient, or carrier. Thecompounds may be added to a carrier in the form of a salt or solvate.For example, in cases where compounds are sufficiently basic or acidicto form stable nontoxic acid or base salts, administration of thecompounds as salts may be appropriate. Examples of pharmaceuticallyacceptable salts are organic acid addition salts formed with acids thatform a physiologically acceptable anion, for example, tosylate,methanesulfonate, acetate, citrate, malonate, tartrate, succinate,benzoate, ascorbate, α-ketoglutarate, and β-glycerophosphate. Suitableinorganic salts may also be formed, including hydrochloride, halide,sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid to provide aphysiologically acceptable ionic compound. Alkali metal (for example,sodium, potassium or lithium) or alkaline earth metal (for example,calcium) salts of carboxylic acids can also be prepared by analogousmethods.

The compounds of the formulas described herein can be formulated aspharmaceutical compositions and administered to a mammalian host, suchas a human patient, in a variety of forms. The forms can be specificallyadapted to a chosen route of enteral administration, e.g., oraladministration, sublingual administration, or rectal administration.

The compounds described herein may be systemically administered incombination with a pharmaceutically acceptable vehicle, such as an inertdiluent or an assimilable edible carrier. For oral administration,compounds can be enclosed in hard or soft shell gelatin capsules,compressed into tablets, or incorporated directly into the food of apatient's diet. Compounds may also be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations typically contain at least 0.1% ofactive compound. The percentage of the compositions and preparations canvary and may conveniently be from about 0.5% to about 60%, about 1% toabout 25%, or about 2% to about 10%, of the weight of a given unitdosage form. The amount of active compound in such therapeuticallyuseful compositions can be such that an effective dosage level can beobtained.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; and a lubricant such as magnesium stearate. A sweeteningagent such as sucrose, fructose, lactose or aspartame; or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring, maybe added. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweetening agent, methyl and propyl parabens as preservatives, a dye andflavoring such as cherry or orange flavor. Any material used inpreparing any unit dosage form should be pharmaceutically acceptable andsubstantially non-toxic in the amounts employed. In addition, the activecompound may be incorporated into sustained-release preparations anddevices.

Solutions of the active compound or its salts can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can be preparedin glycerol, liquid polyethylene glycols, triacetin, or mixturesthereof, or in a pharmaceutically acceptable oil. Under ordinaryconditions of storage and use, preparations may contain a preservativeto prevent the growth of microorganisms.

Pharmaceutical dosage forms include aqueous solutions, dispersions, orsterile powders comprising the active ingredient, optionallyencapsulated in liposomes. The ultimate dosage form should be sterile,fluid and stable under the conditions of manufacture and storage. Aliquid carrier or vehicle can be a solvent or liquid dispersion mediumcomprising, for example, water, ethanol, a polyol (for example,glycerol, propylene glycol, liquid polyethylene glycols, and the like),vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.The proper fluidity can be maintained, for example, by the formation ofliposomes, by the maintenance of the required particle size in the caseof dispersions, or by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialand/or antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, buffers, orsodium chloride. Prolonged absorption of the compositions can be broughtabout by agents capable of delaying absorption, for example, aluminummonostearate and/or gelatin.

Various dosage forms can be prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousother ingredients enumerated above, optionally followed by filtersterilization. Methods of preparation can include vacuum drying andfreeze-drying techniques, which yield a powder of the active ingredientplus any additional desired ingredient present in the solution.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina, and the like. Useful liquidcarriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, orwater-alcohol/glycol blends, in which a compound can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be administered orally or sprayed intothe mouth using a pump-type or aerosol sprayer. Thickeners such assynthetic polymers, fatty acids, fatty acid salts and esters, fattyalcohols, modified celluloses, or modified mineral materials can also beemployed with liquid carriers.

Useful dosages of the compounds described herein can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949 (Borch et al). The amount of a compound, or an activesalt or derivative thereof, required for use in treatment will vary notonly with the particular compound or salt selected but also with theroute of administration, the nature of the condition being treated, andthe age and condition of the patient, and will be ultimately at thediscretion of an attendant physician or clinician.

In general, however, a suitable dose will be in the range of from about0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of bodyweight per day, such as 3 to about 50 mg per kilogram body weight of therecipient per day, preferably in the range of 6 to 90 mg/kg/day, mostpreferably in the range of 15 to 60 mg/kg/day.

The compound is conveniently formulated in unit dosage form; forexample, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form. Inone embodiment, the invention provides a composition comprising acompound of the invention formulated in such a unit dosage form.

The compound can be conveniently administered in a unit dosage form, forexample, containing 5 to 1000 mg/m², conveniently 10 to 750 mg/m², mostconveniently, 50 to 500 mg/m² of active ingredient per unit dosage form.The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

The compounds described herein can be effective anti-bacterial agentsand have higher potency and/or reduced toxicity as compared tovancomycin. Preferably, compounds of the invention are more potent andless toxic than vancomycin, and/or avoid a potential site of catabolicmetabolism encountered with vancomycin, and have a differentpharmacokinetic profile than vancomycin.

The invention provides therapeutic methods of treating bacterialinfections in a mammal, which involve administering to a mammal havingbacterial infection an effective amount of a compound or compositiondescribed herein. A mammal includes a primate, human, rodent, canine,feline, bovine, ovine, equine, swine, caprine, bovine and the like.Bacterial infections refer to any various type of bacterium that causesdebilitating or life-threatening health issues.

The ability of a compound of the invention to treat bacterial infectionsmay be determined by using assays well known to the art. For example,the design of treatment protocols, toxicity evaluation, data analysis,quantification of bacterial cell death, and the biological significanceof the use of bacterial screens are known. In addition, ability of acompound to treat bacterial infections may be determined using theprotocols as described herein.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention.

EXAMPLES Example 1 Materials and Methods

Abbreviations. AUC, area under the curve; CDI, C. difficile infection;DMF, dimethylformamide; DMSO, dimethyl sulfoxide; ESI, electrosprayionization; HRMS, high resolution mass spectrometry; ip,intraperitoneal; iv, intravenous; MIC, minimally-inhibitoryconcentration, MRM, multiple reaction monitoring; MS, mass spectrometry;PK, pharmacokinetic; po, oral; THF, tetrahydrofuran; TLC, thin-layerchromatography; UPLC, ultra-performance liquid chromatography.

Synthesis. All chemicals, reagents, and solvents were used directly aspurchased without further purification. Analytical thin-layerchromatography was performed on silica gel 60 F₂₅₄. For columnchromatography, silica gel 60, 230-400 mesh, 40-63 μm was used. ¹H NMRand ¹³C NMR spectra were recorded on Bruker AVANCE III HD 500spectrometers (Bruker Daltonik, Bremen, Germany) and operated at an ¹Hresonance frequency of 500.13 MHz. Chemical shifts are referenced to theresidual deuterated solvent (e.g., for CDCl₃, δ=7.26 and 77.16 ppm for¹H and ¹³C NMR, respectively) and reported in parts per million (ppm, δ)relative to tetramethylsilane (TMS, δ=0.00 ppm). Coupling constants (J)are reported in Hz, where s=singlet, d=doublet, t, triplet, m=multiplet,br=broad. High-resolution mass spectra were measured using a BrukermicrOTOF/Q2 mass spectrometer in electron spray ionization source (ESI)ionization.

4-(Cyclopentyloxy)benzonitrile (5)

Cyclopentanol (4) (1.54 g, 18 mmol) and 0.72 g NaH were placed at 50 mLtwo-neck reaction flask at 0° C. 4-Fluorobenzenitrile (3) (1.81 g, 15mmol) in 3 mL DMF was added to the above mixture. The reaction mixturewas stirred at 0° C. for 15 minutes and was allowed to come to roomtemperature. The reaction was monitored by TLC and was complete after 2h. The mixture was quenched in iced water and extracted with ethylacetate (3×50 mL). The organic phases were combined and dried overanhydrous Na₂SO₄, followed by filtration and concentration of thefiltrate under reduced pressure. The residue was purified on silica gelchromatography (hexane/ethyl acetate, 25:1) to give compound 5 as acolorless oil (3.06 g, 91%).

¹H NMR (500 MHz, CDCl₃) δ 1.65−1.59 (m, 2H), 1.83−1.75 (m, 4H),1.94−1.90 (m, 2H), 4.77 (t, J=2.5 Hz, 1H), 6.89−6.86 (m, 2H), 7.53−7.50(m, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 24.2, 32.9, 80.1, 103.3, 116.2,119.6, 134.0, 161.7; HRMS [M+H]⁺, calcd for C₁₂H₁₄NO 188.1070; found188.1099.

(Z)-4-(Cyclopentyloxy)-N′-hydroxybenzimidamide (6)

Compound 5 (4.47 g, 23.89 mmol) was dissolved in ethanol (125 mL) in a200-mL reaction flask. NH₂OH (50%, 5.6 mL) was added and the mixture wasstirred at room temperature for 50 h until the reaction was complete asmonitored by TLC. The solvent was evaporated under reduced pressure toafford the product as a white solid (5.26 g, 100%).

¹H NMR (500 MHz, DMSO-d₆) δ 1.58−1.52 (m, 2H), 1.70−1.63 (m, 4H),1.92−1.82 (m, 2H), 4.81−4.78 (m, 1H), 5.70 (s, 2H), 6.87−6.84 (m, 2H),7.58−7.55 (m, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 24.3, 32.9, 79.3, 115.5,126.0, 127.4, 151.3, 158.9; HRMS [M+H]⁺, calcd for C₁₂H₁₇N₂O₂ 221.1824;found 211.1283.

5-(4-(Allyloxy)phenyl)-3-(4-(cyclopentyloxy)phenyl)-1,2,4-oxadiazole (8)

Compound 6 (0.62 g, 3.5 mmol) and 1,1′-carbonyldiimidazole (0.53 g, 3.25mmol) were dissolved in 4 mL DMF at 50° C. The mixture was stirred for15 minutes and compound 7 (0.55 g, 2.5 mmol) in 15 mL THF was added. Thetemperature was increased to 100° C. and the reaction was complete after14 h. The reaction was quenched with saturated NH₄Cl solution and wasextracted with ethyl acetate (25 mL, 3×). The organic phases werecombined, dried over anhydrous Na₂SO₄, filtered, and concentrated underreduced pressure. The crude material was purified on silica gelchromatography (hexane/ethyl acetate, 50:1) to give the title compound 8as a viscous oil (0.70 g, 78%).

¹H NMR (500 MHz, CDCl₃) δ 1.66−1.60 (m, 2H), 1.97−1.75 (m, 6H),4.62−4.61 (m, 2H), 4.84−4.82 (m, 1H), 5.34−5.32 (m, 1H), 5.46−5.42 (m,1H), 6.09−6.02 (m, 1H), 6.96 (d, J=9.0 Hz, 2H), 7.03 (d, J=9.0 Hz, 2H),8.06 (d, J=9.0 Hz, 2H), 8.13 (d, J=9.0 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃)δ 24.3, 33.1, 69.2, 79.7, 115.4, 115.9, 117.4, 118.4, 119.3, 129.2,130.2, 132.7, 160.7, 162.3, 168.8, 175.4; HRMS [M+H]⁺, calcd forC₂₂H₂₃N₂O₃ 363.1703; found 363.1709.

4-(3-(4-(Cyclopentyloxy)phenyl)-1,2,4-oxadiazol-5-yl)phenol (2)

A 50-mL flask was charged with Pd(PPh₃)₄ (19 mg, 0.016 mmol) andcompound 6 (0.30 g, 0.82 mmol) in 12 mL THF under Ar atmosphere at roomtemperature. The mixture was stirred for 5 minutes and NaBH₄ (47 mg,1.23 mmol) was added. The reaction mixture was stirred for 1.5 h,quenched with aqueous HCl (1M, 3 mL), and extracted with ethyl acetate(3×20 mL). The organic phases were combined and dried over anhydrousNa₂SO₄, followed by filtration and concentration under reduced pressure.The residue was purified on silica gel chromatography (hexane/ethylacetate, 6:1) to afford 2 as white solid (0.23 g, 87%).

¹H NMR (500 MHz, DMSO-d₆) δ 1.61−1.55 (m, 2H), 1.74−1.66 (m, 4H),1.98−1.90 (m, 2H), 4.91−4.83 (m, 1H), 6.99−6.96 (m, 2H), 7.07−7.04 (m,2H), 8.00−7.94 (m, 4H); ¹³C NMR (125 MHz, DMSO-d₆) δ 24.3, 32.9, 79.7,114.9, 116.5, 116.9, 118.9, 129.4, 130.7, 160.8, 162.6, 168.4, 175.8;HRMS [M+H]⁺, calcd for C₁₉H₁₉N₂O₃ 323.1390; found 323.1419.

Antibiotics. Vancomycin and metronidazole were purchased from SigmaAldrich (St. Louis, Mo.) and fidaxomicin was obtained from BOC Sciences(Shirley, N.Y.).

Bacterial strains. The strains used in the study were obtained from ATCC(Manassas, Va.) and BEI Resources (Manassas, Va.). All the strains werecultured and stored according to the supplier instructions.

Minimally-inhibitory concentrations. MICS for C. difficile strains andthe common gut bacteria were determined using broth microdilutiontechniques as reported earlier using brucella broth supplemented withhemin and vitamin K or supplemented BHIS broth (Babakhani et al., JAntimicrob Chemother 2013, 68(3), 515). Lactobacillus MRS broth was usedfor lactobacillus strains. The test compounds were added in 2-foldserial dilutions and the bacteria were added to a final concentration of5×10⁵ cfu/mL. All incubations unless specified otherwise were carriedout at 24 or 48 h at 37° C. in an anaerobic chamber (Whitley DG250workstation, Microbiology International, Frederick, Md.).Corynebacterium and lactobacillus species were incubated aerobically.

Inhibition of spores and toxins in exponential phase cells. C. difficileATCC43255 was used for this assay. Colonies from an overnight culturewere suspended in supplemented BHIS broth to a final concentration of˜10⁸ cfu/mL. Serial 2-fold dilutions of the test compounds were preparedstarting at 2- or 4-fold below the MIC and 2- or 4-fold above the MIC ofthe compound to a final volume of 10 mL and the bacteria were added toobtain a final concentration of 5×10⁵ cfu/mL. The tubes were incubatedanaerobically for 3 days. A 100-μl aliquot of the supernatant wasremoved every 24 h and stored at −80° C. for toxin analysis. Forquantification of viable cells and spores additional 100-μL aliquotswere removed and plated. Spores were quantified as reported earlierusing ethanol treatment (Goldstein et al, Anaerobe 2010, 16(3), 220).

Toxin A and B detection. Total toxin A and B levels in the culturesupernatant were measured using a commercial enzyme linked immunosorbentassay (ELISA) kit (Premier Toxins A and B, Meridian Bioscience Inc.,Cincinnati, Ohio) according to the manufacturer's recommendations. Datawere read at A₄₅₀ using ELISA plate reader (Epoch, BioTek instrumentsInc., Winooski, Vt.).

Macromolecular synthesis assays. The method of Mathur et al (JAntimicrob Chemother 2011, 66(5), 1087) was used with a fewmodifications. [2,8-³H]-adenine (40 Ci/mmol), 1-[3,4,5-³H(N)]-leucine(120 Ci/mmol), and N-acetyl-d-[6-³H]-glucosamine (30 Ci/mmol) werepurchased from Perkin Elmer (Waltham, Mass.). Ciproflaxin, linezolid,and oxacillin, were used as positive controls for DNA, protein, andpeptidoglycan, respectively. Ciprofloxacin and oxacillin were purchasedfrom Sigma-Aldrich (St. Louis, Mo.), linezolid was obtained fromAmplaChem Inc. (Carmel, Ind.). Radiolabeled precursors (finalconcentration of 1.0 μCi/mL for DNA and peptidoglycan and 2.0 μCi/mL forprotein) were incubated with logarithmically growing C. difficile ATCC43255 at 37° C., followed by addition of oxadiazole 2 at ½, 1, or 2×MIC, with further incubation for 180 min. Aliquots were taken every 30min for radioactive counting and viability. Optimal incubation timeswere 60 min for DNA, 180 min for protein, and 120 min for peptidoglycan.

Animals. Female C57Bl/6J (8-9 weeks old) were purchased from EnvigoCorp. (Huntington, United Kingdom) for the C. diffcile study. Female ICRmice (6-8 weeks old, ˜20-g body weight) were used for the PK studies andpurchased from Harlan Laboratories, Inc. (Indianapolis, Ind.). Mice weregiven Teklad 2019 Extruded Rodent Diet and water ad libitum. Mice weremaintained in polycarbonate shoebox cages with ¼ in. corncob (TheAndersons Inc., Maumee, Ohio) and Alpha-dri (Sheperd Specialty Papers,Inc., Richland, Mich.) bedding under 12-h light/12-h dark cycle at 72±2°F. All procedures involving vertebrate animals were approved by theInstitutional Animal Care and Use Committee at the University of NotreDame.

Pharmacokinetic (PK) Studies. Oxadiazole 2 was dissolved in 10% DMSO/25%Tween-80/65% water at a concentration of 5 mg/mL. Mice (n=3 per timepoint) were administered 100 μL (equivalent to 20 mg/kg) po by gastricintubation or intravenously (iv) by tail vein injection. Terminal bloodwas collected by cardiac puncture with sodium heparin at the followingtime points: 0.5, 1, 2, 3, 4, 6, 9, 24, 36 h for po administration and2, 5, 10, 20, 40 min and 1, 2, 3, 4, 8, 24 h for iv administration.Whole blood was centrifuged at 1000 g for 10 min to obtain plasma.Aliquots of the plasma samples (50 μL) were analyzed the day ofcollection; the remaining plasma was stored at −80° C.

Plasma samples were analyzed by ultra-performance liquid chromatography(UPLC, Waters Corp., Milford, Mass.) coupled to a triple quadrupole massspectrometer (TQD, Waters) operating in multiple reaction monitoring(MRM) mode. A standard curve of oxadiazole 2 was prepared in 50 μL ofblank mouse plasma. Protein was precipitated by addition of 150 μL ofacetonitrile containing internal standard. Samples were centrifuged at22,000 g for 15 minutes, and the supernatants were analyzed byUPLC-MS/MS-MRM. Acquisition parameters were as follows: Supelco AscentisC18 column (3 μm particle size, 10 cm×2.1 mm; Sigma-Aldrich, St. Louis,Mo.), electrospray ionization positive mode (ESI+), flow rate 0.5mL/min, capillary voltage 4 kV, cone voltage 30 V, and collision voltage25 V. The solvent program was as follows: 95% A-5% B for 0.25 min,0.75-min linear gradient to 5% A-95% B, hold for 4 min, where A is 0.1%formic acid/water and B is 0.1% formic acid/acetonitrile. The method waslinear between 0 μM and 20 μM (R² values 0.98-0.99). MRM transitionswere 323.0→121.1 for oxadiazole 2 and 401.1→122.8 for the internalstandard. Peak areas for oxadiazole 2 and the internal standard werecalculated using Waters MassLynx software. A standard curve of peak arearatio to the internal standard plotted against standard concentrationwas generated from which concentrations of oxadiazole 2 in the plasmasamples from the PK studies were determined using regression parameters.

Phoenix WinNonlin 6.3 (Certara LP, St Louis, Mo.) noncompartmentalanalysis using uniform weighing was used to calculate the area under thecurve (AUC), clearance (CL), volume of distribution (Vd), and terminalhalf-life. Half-lives were estimated from the linear portion of theinitial or terminal phases of the concentration-time data by linearregression, where the slope of the line was the rate constant k andt_(1/2)=ln 2/k.

Toxicity of Oxadiazole 2. Female uninfected C57Bl/6 mice (n=3) weregiven multiple po doses of oxadiazole 2 at 40 mg/kg once a day for 5days. The oxadiazole was dissolved in 5% DMSO/25% Tween-80/70% water.Mice were clinically observed and body weights were recorded daily.Feces were collected daily and analyzed for concentrations of theoxadiazole by UPLC with MRM detection (see Table 3).

C. difficile Mouse Model. The model developed by Chen et al(Gastroenterology 2008, 135(6), 1984) was used with a few modifications.Mice (n=10/group) were treated with a mixture of kanamycin (0.4 mg/mL),gentamicin (0.035 mg/mL), colistin (850 U/mL), metronidazole (0.215mg/mL), and vancomycin (0.045 mg/mL) added to the drinking water andsupplemented with 5% sucrose for five days. After antibiotic treatment,the mice were given autoclaved water for 2 days. The following day, micewere administered a single 10 mg/kg intraperitoneal (ip) dose ofclindamycin. One day later (day 0), the mice are infected with 10⁴ cfuof C. difficile strain VPI 10463 (ATCC 43255) intragastrically. One hourafter infection, vancomycin (50 mg/kg/day oral (po)) or oxadiazole 2 (20mg/kg/day po) was given for 5 days. A negative vehicle control will beincluded. Survival was recorded daily for 25 days and body weights wererecorded 5 times a week. This study was conducted in duplicates.

A second study was conducted with oxadiazole 2 given po at 20 or 40mg/kg for 5 days and vancomycin administered at 50 mg/kg po for 5 days.Fecal samples were collected on days 3, 7, and 10 for C. difficile sporeand toxin evaluation.

Statistical Analyses. Data were analyzed for statistical significanceusing the Student t-test (Excel) using a two-tail distribution andunequal variance.

Summary. Novel oxadiazoles with activity against C. difficile aredisclosed. Oxadiazole 2 has similar activity against NAP1/027 strainsBAA-1870 and BAA-1803 as vancomycin; it inhibits not only vegetativecells, but spore and toxin production. The compound inhibitspeptidoglycan synthesis, it is poorly absorbed, and it attains highconcentrations in the gut. Oxadiazole 2 is well tolerated in mice andshows efficacy better than vancomycin in a mouse model of recurrent CDI.

Example 2 Pharmaceutical Dosage Forms

The following formulations illustrate representative pharmaceuticaldosage forms that may be used for the therapeutic or prophylacticadministration of a compound of a formula described herein, a compoundspecifically disclosed herein, or a pharmaceutically acceptable salt orsolvate thereof (hereinafter referred to as ‘Compound X’):

(i) Tablet 1 mg/tablet ‘Compound X’ 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0

(ii) Tablet 2 mg/tablet ‘Compound X’ 20.0 Microcrystalline cellulose410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0500.0

(iii) Capsule mg/capsule ‘Compound X’ 10.0 Colloidal silicon dioxide 1.5Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0

(iv) Injection 1 (1 mg/mL) mg/mL ‘Compound X’ (free acid form) 1.0Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodiumchloride 4.5 1.0N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(v) Injection 2 (10 mg/mL) mg/mL ‘Compound X’ (free acid form) 10.0Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethyleneglycol 400 200.0 0.1N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(vi) Aerosol mg/can ‘Compound X’ 20 Oleic acid 10Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000Dichlorotetrafluoroethane 5,000

(vii) Topical Gel 1 wt. % ‘Compound X’   5% Carbomer 934 1.25%Triethanolamine q.s. (pH adjustment to 5-7) Methyl paraben  0.2%Purified water q.s. to 100 g

(viii) Topical Gel 2 wt. % ‘Compound X’   5% Methylcellulose   2% Methylparaben  0.2% Propyl paraben 0.02% Purified water q.s. to 100 g

(ix) Topical Ointment wt. % ‘Compound X’   5% Propylene glycol   1%Anhydrous ointment base  40% Polysorbate 80   2% Methyl paraben 0.2%Purified water q.s. to 100 g

(x) Topical Cream 1 wt. % ‘Compound X’  5% White bees wax 10% Liquidparaffin 30% Benzyl alcohol  5% Purified water q.s. to 100 g

(xi) Topical Cream 2 wt. % ‘Compound X’   5% Stearic acid  10% Glycerylmonostearate   3% Polyoxyethylene stearyl ether   3% Sorbitol   5%Isopropyl palmitate   2% Methyl Paraben 0.2% Purified water q.s. to 100g

These formulations may be prepared by conventional procedures well knownin the pharmaceutical art. It will be appreciated that the abovepharmaceutical compositions may be varied according to well-knownpharmaceutical techniques to accommodate differing amounts and types ofactive ingredient ‘Compound X’. Aerosol formulation (vi) may be used inconjunction with a standard, metered dose aerosol dispenser.Additionally, the specific ingredients and proportions are forillustrative purposes. Ingredients may be exchanged for suitableequivalents and proportions may be varied, according to the desiredproperties of the dosage form of interest.

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, such embodiments are onlyillustrative and do not limit the scope of the invention. Changes andmodifications can be made in accordance with ordinary skill in the artwithout departing from the invention in its broader aspects as definedin the following claims.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Nolimitations inconsistent with this disclosure are to be understoodtherefrom. The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

What is claimed is:
 1. A method of treating a Clostridium difficilebacterial infection in a gastrointestinal tract of a subject comprisingadministering to a subject in need thereof an effective amount ofcompound 2:

or a salt thereof; wherein the Clostridium difficile bacterial infectionis located in the gastrointestinal tract of the subject and less than 30percent of compound 2 is absorbed by the gastrointestinal tract, therebytreating the Clostridium difficile infection.
 2. The method of claim 1further comprising administering an agent capable of delaying absorptionof the compound.